On the cDNA's for two types of rat pancreatic secretory trypsin inhibitor

Two types of cDNA, which code for the two types of rat pancreatic secretory trypsin inhibitors (PSTIs), were cloned and sequenced. Both predicted amino acid sequences consisting of 79 amino acids, with the secretion signal peptide consisting of 18 and 23 amino acids for PSTI-I and PSTI-II, respectively. The nucleotide sequences were 91% homologous between the two cDNAs, but 68% and 65% homologous, respectively, when compared with human PSTI cDNA. Northern blot analyses showed that PSTI-I is expressed in the pancreas, whereas PSTI-II is expressed in the pancreas and the liver using the same promoter. Southern blot analyses suggested that both PSTI-I and PSTI-II genes are single copy genes per haploid genome. Duplication of rat PSTI gene seems to have occurred recently, after the divergence of humans and rats.     

Expression of human pancreatic secretory trypsin inhibitor in Saccharomyces cerevisiae

Human pancreatic secretory trypsin inhibitor (PSTI) cDNA was expressed in Saccharomyces cerevisiae using the yeast acid phosphatase PHO5 promoter. The product encoded by the PSTI-coding cDNA was correctly processed in yeast cells, and the PSTI molecules were efficiently secreted into the medium. The amino acid composition and the N-terminal amino acid sequence of the secreted PSTI molecules were identical to those of the authentic PSTI polypeptides from human pancreas, and the product exhibited trypsin-inhibitory activity.     

Cloning and characterization of the human colipase cDNA

Pancreatic lipase hydrolyzes dietary triglycerides to monoglycerides and fatty acids. In the presence of bile salts, the activity of pancreatic lipase is markedly decreased. The activity can be restored by the addition of colipase, a low molecular weight protein secreted by the pancreas. The action of pancreatic lipase in the gut lumen is dependent upon its interaction with colipase. As a first step in elucidating the molecular events governing the interaction of lipase and colipase with each other and with fatty acids, a cDNA encoding human colipase was isolated from a lambda gt11 cDNA library with a rabbit polyclonal anti-human colipase antibody. The full-length 525 bp cDNA contained an open reading frame encoding 112 amino acids, including a 17 amino acid signal peptide. The predicted protein sequence contains 100% of the published protein sequence for human colipase determined by chemical methods, but predicts the presence of five additional NH2-terminal amino acids and four additional COOH-terminal amino acids. Comparison of the predicted protein sequence with the known sequences of colipase from other species reveals regions of extensive identity. In vitro translation of mRNA transcribed from the cDNA gave a protein of the expected molecular size that was processed by pancreatic microsomal membranes. Sequence analysis of the in vitro translation product after processing demonstrated signal peptide cleavage and the presence of a human procolipase, as exists in the pig and horse colipases. DNA blot analysis was consistent with the presence of a single gene for colipase. RNA blot analysis demonstrated tissue-specific expression of colipase mRNA in the pancreas. Thus, we report, for the first time, a cDNA for colipase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kirrel2, a novel immunoglobulin superfamily gene expressed primarily in beta cells of the pancreatic islets

A novel immunoglobulin superfamily (Igsf) protein gene was discovered by computational analysis of human draft genomic DNA, and multiple cDNA clones were obtained. The protein encoded by this gene contains five Ig domains, one transmembrane domain, and an intracellular domain. It has significant similarity with several known Igsf proteins, including Drosophila RST (irregular chiasm C-roughest) protein and mammalian KIRREL (kin of irregular chiasm C-roughest), NEPH1, and NPHS1 (nephrin) proteins. All these proteins have multiple Ig domains, possess properties of cell adhesion molecules, and play important roles in organ development. RT-PCR and Northern blot results indicate this gene is predominantly expressed in pancreas, and public sequence databases indicate there is also expression in the nervous system. We have named this gene Kirrel2 (kin of irregular chiasm-like 2), to reflect its similarity to irregular chiasm C-roughest and Kirrel. Four splice forms of Kirrel2 were observed, including two that we cloned from pancreas mRNA as well as two GenBank entries, one from the brain and one from a retinoblastoma cell line. A partial cDNA clone of the mouse orthologue was obtained by RT-PCR from mouse brain, and the inferred protein sequence has 85% sequence identity to the human protein. Immunohistochemical staining results indicate that the KIRREL2 protein is conserved from rodents to primates, and it is highly expressed in pancreatic islets. RT-PCR results on mouse pancreatic cell lines indicate that expression in the pancreas is restricted to beta cells. Thus, KIRREL2 protein is a beta-cell-expressed Ig domain protein and may be involved in pancreas development or beta cell function.     

Immunohistochemical localization of pancreatic secretory trypsin inhibitor in fetal and adult pancreatic and extrapancreatic tissues

Pancreatic secretory trypsin inhibitor (PSTI) has been thought to be only a secretory trypsin inhibitor of human pancreas, but the serum content of immunoreactive PSTI is elevated without pancreatic disease. Using the peroxidase-antiperoxidase method, immunoreactive cells for PSTI were found in human pancreas, stomach, duodenum, appendix, colon and urinary tract of both fetus and adult, adult gall bladder, and fetal lung. PSTI-immunoreactive cells were identified in fetal pancreas at the tenth gestational week, and in extrapancreatic tissues at the sixteenth (gastrointestinal and urinary tract) and twentieth weeks (lung). PSTI-immunoreactive cells of fetal lung were present in neuroepithelial bodies. Strongly positive cells in fetal duodenum were argyrophilic and resembled endocrine cells. Immunohistochemical study was also performed on tissues associated with inflammatory diseases of gastrointestinal tract. The distribution pattern of immunoreactive cells in the stomach varied in accordance with chronic gastritis. Immunoreactive cells were also found in endocrine micro-nests and in a carcinoid tumor associated with fundic gastritis. These results suggest that PSTI may play some physiological role other than secretory trypsin inhibition of the pancreas.     

Development of human pancreas

The developmental sequence of human pancreatic secretory proteins has not previously been studied in detail. We applied immunohistochemistry to study 20 fetal and neonatal pancreas' (8th to 39th gestational weeks) using antisera against the following pancreatic secretory proteins: pancreatic secretory trypsin inhibitor (PSTI), serine proteinases (trypsin, chymotrypsin, and elastase I), and amylase. PSTI was first detected in developing buds of the pancreas during the 8th gestational week, and proteinases were observed in acinar cells during the 14th week of gestation. Immunoreactivity for both PSTI and proteinases was found in most acinar cells soon after their appearance. Immunoreactivity for amylase could not be detected in fetal or neonatal pancreas tissue. PSTI was also found in developing islets during the 14th gestational week, but the number of immunoreactive cells had decreased by term. Cells positive for serine proteinases were occasionally in contact with islets in second-trimester fetuses. In discussing these results, we give particular attention to the nonparallel appearance of secretory products in the fetal pancreas, and the significance of cells immunoreactive for secretory proteins in endocrine islets.     

Isolation of a cDNA encoding a human serum marker for acute pancreatitis. Identification of pancreas-specific protein as pancreatic procarboxypeptidase B.

A human pancreas-specific protein (PASP), previously characterized as a serum marker for acute pancreatitis and pancreatic graft rejection, has been identified as pancreatic procarboxypeptidase B (PCPB). cDNAs encoding PASP/PCPB were isolated from a human pancreas cDNA library using a combination of nucleic acid hybridization screening and immunoscreening with antisera raised against native PASP. The deduced amino acid sequence of PASP/PCPB cDNA predicts the translation of a 416-amino acid preproenzyme with a 15-amino acid signal/leader peptide and a 95-amino acid activation peptide. The proenzyme portion of this protein has 76% identity with rat PCPB and 84% identity with bovine carboxypeptidase B. DNA and RNA blot analyses indicate that human PCPB mRNA (1,400 nucleotides) is transcribed from a single locus in the human genome in a tissue-specific fashion. N-terminal sequencing of native PASP and the specific immunoreactivity of bacterially expressed PASP/PCPB with native PASP antibodies confirm the identification of PASP as human pancreatic PCPB.     

Production of recombinant human pancreatic secretory trypsin inhibitor by Escherichia coli

A synthetic gene for human pancreatic secretory trypsin inhibitor (PSTI) was fused to the coding sequence for the amino-terminal 135 amino acid residues of human interferon-gamma (IFN-gamma) by interposing a methionine codon sequence, and the resulting hybrid gene was efficiently expressed in Escherichia coli cells. Recombinant human PSTI (rHu-PSTI) was separated from the IFN-gamma/PSTI fused protein by cleavage at the methionine residue with cyanogen bromide. Finally, rHu-PSTI was purified by affinity chromatography on a bovine trypsin-CH-Sepharose 4B column. The amino acid composition, partial amino-terminal sequence, disulfide formation, human trypsin inhibitory activity, and immunoreactivity against rabbit anti-human PSTI serum of rHu-PSTI corresponded to those of the natural form.     

Assignment of human pancreatic lipase gene (PNLIP) to chromosome 10q24-q26.

Human pancreatic lipase (EC 3.1.1.3) is a 56-kDa protein secreted by the acinar pancreas and is essential for the hydrolysis and absorption of long-chain triglyceride fatty acids in the intestine. In vivo, the 12-kDa protein cofactor, colipase, is required to anchor lipase to the surface of lipid micelles, counteracting the destabilizing influence of bile salts. Southern blot analysis, using a pancreatic lipase cDNA to probe DNA from mouse-human somatic cell hybrids, indicated that the pancreatic lipase gene (PNLIP) resides on human chromosome 10. In situ hybridization to human metaphase chromosomes confirmed the cell hybrid results and further localized the gene to the 10q24-qter region with the strongest peak at q26.1.     

Exons of the human pancreatic polypeptide gene define functional domains of the precursor

Pancreatic polypeptide is a 36-amino acid peptide which inhibits pancreatic exocrine function. We have previously determined from the nucleotide sequence of a cDNA that pancreatic polypeptide is derived from a 95-amino acid precursor, prepropancreatic polypeptide. Pulse-chase studies have suggested that the precursor is cleaved to produce three peptides: pancreatic polypeptide, an icosapeptide, and a smaller peptide. In the present study, we have used the cloned cDNA as a hybridization probe to isolate the pancreatic polypeptide gene from a human bacteriophage genomic library. The nucleotide sequence of 2.8 kilobases of DNA representing the entire human pancreatic polypeptide gene was determined. The gene contains four exons and three introns. Exon 1 encodes the 5'-untranslated region of the mRNA, exon 2 encodes the signal sequence and the sequence of pancreatic polypeptide, exon 3 encodes the icosapeptide, and exon 4 encodes a carboxyl-terminal heptapeptide and the 3'-untranslated region of the mRNA. By Southern blot analysis, the gene detected in a pancreatic polypeptide-producing islet cell tumor was indistinguishable from that in normal human leukocytes. The structure of the human pancreatic polypeptide gene is consistent with the hypothesis that prepropancreatic polypeptide generates three distinct peptides, each encoded by a separate exon. Increased expression of pancreatic polypeptide in the islet cell tumor does not appear to be correlated with major alterations in pancreatic polypeptide gene structure.     

Identification of a novel class of elastase isozyme, human pancreatic elastase III, by cDNA and genomic gene cloning

By using porcine elastase I cDNA as a probe, we have isolated two different but closely related cDNAs encoding elastase-like proteases from a human pancreatic cDNA library. The amino acid sequences deduced from the cloned cDNA sequences showed 56-61% identity with those of both pancreatic elastases I and II, similar to the homology between elastases I and II. The active form of the elastase-like proteases appeared to be composed of 242 amino acids and preceded by a signal peptide and propeptide of 28 amino acids. Dot blot analysis of various tissue mRNAs demonstrated that the genes for the cloned cDNAs are expressed at a high level only in the pancreas. In addition, sequence analysis of the cloned genomic genes corresponding to one of the cDNAs showed that they are members of the elastase gene family. These results indicate that the two enzymes encoded by the cDNAs should be classified into a third class of elastase isozyme. Therefore, we designated them as human pancreatic elastases IIIA and IIIB. They strongly resembled cholesterol-binding pancreatic protease, suggesting that they may possess not only a digestive function but also function(s) related to cholesterol metabolism or transport in the intestine.     

Isolation and sequence of a novel amphibian pancreatic chitinase

An approximately 60-kDa protein with chitinase activity was purified from the pancreas of the toad Bufo japonicus. Its specific activity was 4.5 times higher than that of a commercial bacterial chitinase in fragmenting crab shell chitin, and its optimal pH was approximately 6.0. A cDNA clone encoding a protein consisting of 488 amino acid residues, including part of the peptide sequence determined from the isolated protein, was obtained from a toad pancreas cDNA library. The deduced amino acid sequence indicated that the protein contained regions with high homology to those present in chitinases from different species, with the amino acid residues for the chitinase activity and the chitin-binding ability being completely conserved. We designate the protein as toad pancreatic chitinase (tPCase). Northern blot analysis revealed the mRNA of this enzyme to be expressed exclusively in the pancreas. Toad PCase is the first amphibian chitinase to be identified as well as the first pancreatic chitinase identified in a vertebrate.     

Dickkopf-3 is expressed in a subset of adult human pancreatic beta cells

The Dickkopf (Dkk) gene family of secretory modulators of canonical Wnt/beta catenin signals is involved in the control of stem cell proliferation, homeostasis and differentiation. Bioinformatic data on dkk-1/3 gene expression, indicating high expression levels in the human pancreas, led us to analyze these two proteins in adult human pancreatic tissue. Dkk-1/3 mRNA levels and protein distribution were analyzed in isolated human islets vs. the exocrine/ductal pancreatic cells and in paraffin sections of adult human pancreata. Using real time PCR only lowest amounts of dkk-1 mRNA were detectable in the endocrine fractions. Immunohistochemistry did not reveal any Dkk-1 protein in adult human pancreatic tissue. Interestingly, Dkk-3 mRNA and protein were clearly present in adult human pancreatic islets. Messenger RNA levels for Dkk-3 were significantly higher in isolated islets as compared to the exocrine/ductal fraction. Co-staining with an antibody against insulin identified the beta cells of the pancreas as the Dkk-3-positive cells. Notably, only a subset of beta cells contained Dkk-3. As shown by western blot analysis Dkk-3 seems to be proteolytically processed in beta cells. To our knowledge, this is the first study describing a molecule with which the pool of pancreatic beta cells can be further subdivided. Future studies will show whether this sub-classification of beta cells translates into functional differences.     

GPR40 gene expression in human pancreas and insulinoma

To assess gene expression of a membrane-bound G-protein-coupled fatty acid receptor, GPR40, in the human pancreas and islet cell tumors obtained at surgery were analyzed. The mRNA level of the GPR40 gene in isolated pancreatic islets was approximately 20-fold higher than that in the pancreas, and the level was comparable to or rather higher than that of the sulfonylurea receptor 1 gene, which is known to be expressed abundantly in human pancreatic beta cells. A large amount of GPR40 mRNA was detected in tissue extracts from two cases of insulinoma, whereas the expression was undetectable in glucagonoma or gastrinoma. The present study demonstrates that GPR40 mRNA is expressed predominantly in pancreatic islets in humans and that GPR40 mRNA is expressed solely in human insulinoma among islet cell tumors. These results indicate that GPR40 is probably expressed in pancreatic beta cells in the human pancreas.     

Molecular characterization of a family of ligands for eph-related tyrosine kinase receptors.

A family of tyrosine kinase receptors related to the product of the eph gene has been described recently. One of these receptors, elk, has been shown to be expressed only in brain and testes. Using a direct expression cloning technique, a ligand for the elk receptor has been isolated by screening a human placenta cDNA library with a fusion protein containing the extracellular domain of the receptor. This isolated cDNA encodes a transmembrane protein. While the sequence of the ligand cDNA is unique, it is related to a previously described sequence known as B61. Northern blot analysis of human tissue mRNA showed that the elk ligand's mRNA is 3.5 kb long and is found in placenta, heart, lung, liver, skeletal muscle, kidney and pancreas. Southern blot analysis showed that the gene is highly conserved in a wide variety of species. Both elk ligand and B61 mRNAs are inducible by tumour necrosis factor in human umbilical vein endothelial cells. In addition, both proteins show promiscuity in binding to the elk and the related hek receptors. Since these two ligand sequences are similar, and since elk and hek are members of a larger family of eph-related receptor molecules, we refer to these ligands as LERKs (ligands for eph-related kinases).